This project will continue our studies on the soluble molecules produced by T cells which regulate T cell immunity in contact hypersensitivity to 2,4- dinitrofluorobenzene (DNFB) in mice. We have established a large panel of cloned suppressor T cell (Ts) hybridomas which produce a DNP-specific, class I MHC-restricted suppressor molecule. These Ts hybridomas have rearranged TcR alpha and beta chain genes and express a 80-90 kd alpha/beta heterodimer TcR. The suppressor molecule is a 100-11- kd heterodimer molecule and consists of a nonantigen-binding (NAgB) chain which expresses vbeta and cbeta determinants and an antigen-binding (AgB) chain which expresses Calpha determinants. The Nagb chain dictates MHC restriction and the AgB chain dictates antigen specificity. By these parameters the suppressor molecule is a soluble analogue of the TcR. This proposal will provide more definitive information concerning the biochemical properties of the suppressor molecule, its mechanism of action and its relationship to the TcR. Mechanisms will be studied by determining the effect of the suppressor molecule on various parameters of lymphokine production by DNFB- immune T cells in vitro. Studies will also be done to determine if the suppressor molecule is derived from a glycosyl-phosphatidylinositol (GPI)- linked form of the TcR. Ts hybridomas or cells expressing an engineered form of a GPI-linked TcR will be treated with phospholipase C and the supernatants tested for suppressor activity. Genes which encode the receptor and suppressor molecule will be cloned and sequenced and transfected into a panel of TcR alpha or beta chain deleted variants to restore receptor expression and suppressor molecule production. Finally, the suppressor molecule will be isolated from high titered bioreactor supernatants for biochemical characterization, sequence and crystalographic analysis. Results from these studies will enhance our understanding of how Ts cells function, how they regulate T cell-mediated immune responses and how the TcR interacts with antigen/MHC complexes. In addition, these results may provide important information useful for the development of therapeutic molecules which may be used for the antigen-specific regulation of debilitating T cell-mediated autoimmune disease